Experiment 1: Recovery of ASFV from yard soil on macrophages, a first pilot experiment
In a pilot experiment, 5 g of yard soil were spiked with 400 µL of infectious blood at a titer of 7.25 log10HAD50/mL and stored at 4°C or 25°C. In this study, blood and soil were tested at time points 0, 3, 6, 24, 48, and 72 h. First, 5 ml of RPMI-1640 cell culture medium (Thermo Fisher Scientific, Schwerte, Germany) with 10% fetal bovine serum (FBS) and 2% antibiotics (Gibco Penicillin-Streptomycin mix, 10000 U/ml; Thermo Fisher Scientific) was added to the inoculated soil. Then the soil was agitated in the media by vortexing for 45 seconds (see Figure 2 for all steps). Next, soil and media were sonicated for 45 seconds at 4°C with the settings: duty cycle 40%, output 3.5 with a Branson Sonifier 450 (Heinemann Ultraschall- und Labortechnik; Schwäbisch Gmünd, Germany). After sonication, the soil suspension was centrifuged for 30 minutes at 2,500 x g at 4°C. The supernatant was poured over a coffee filter, pushed through a 0.45 µm syringe filter (Millex Filter Units; Merck Millipore Ltd., Tullagreen, Ireland) and the filtrate was stored at -80°C prior to titration.